Neutrophils are the primary cellular component of innate immunity. To prevent unwanted damage to normal tissue, circulating neutrophils are normally poorly responsive to extracellular stimuli. However, to allow appropriate responses to invading bacteria or other inflammatory stimuli, neutrophils undergo a series of phenotypic changes. First, neutrophils are converted from benign cells, capable of circulating without inducing tissue injury, to cells that are primed for an enhanced response. Second, the primed neutrophils are activated to migrate into tissue and generate and release toxic agents capable of killing bacteria or injuring normal cells.
For example, during neutrophil clearance of invading microorganisms, the neutrophils undergo a step-wise conversion from quiescent circulating cells to activated cells capable of producing large quantities of reactive oxygen species (ROS) and releasing bactericidal proteins (1). Neutrophil priming is an intermediate step in this activation process, whereby exposure to pro-inflammatory cytokines and chemokines; such as tumor necrosis factor (TNF)-α, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-8, and platelet activating factor (PAF); enhance neutrophil responses, including the generation of ROS, to a second stimulus, such as the bacterial wall component N-formyl-methionyl-leucyl-phenylalanine (fMLP) (3).
The enzyme responsible for ROS production in neutrophils is the NADPH oxidase; a multicomponent enzyme with components located in the plasma membrane, in the cytosol, and in the membranes of all neutrophil granule subsets (2). The membrane component of NADPH oxidase is a cytochrome b558, a heterodimer composed of gp22phox and gp91phox, and the cytosolic components are p47phox, p67phox, p40phox and the small G-protein Rac2 (3). The production of ROS is normally tightly regulated. However, excessive or inappropriate ROS generation due to enhanced neutrophil priming and activation can lead to injury to normal tissue.
Evidence indicates that neutrophil priming and activation results, at least in part, from exocytosis of intracellular granules from neutrophils. Priming agents, such as TNF-α and PAF, induce exocytosis of neutrophil granules and result in increased plasma membrane expression of gp91phox and gp22phox (2, 3). It has recently been demonstrated that all granules contain gp91phox and gp22phox in their membranes, indicating that exocytosis of each of the neutrophil granules enhances this plasma membrane expression (4). Additionally, it was shown that p38 MAPK regulated neutrophil granule exocytosis in response to TNF-α and lipopolysaccharide (LPS) (5), providing an explanation for the previously recognized role of p38 MAPK in neutrophil priming (6, 7).
Priming enhances the ability of neutrophils to produce ROS and to kill bacteria. However, excessive or inappropriate activation of neutrophils and enhanced generation of ROS also contributes to tissue damage, such as that seen in ischemia-reperfusion injury (I/R) of the heart, brain, and kidneys; anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; rheumatoid arthritis; acute glomerulonephritis; the lung injury of sepsis; and other disorders involving neutrophil-mediated inflammatory processes. Indeed, neutrophils have been shown to mediate damage to postcapillary venules during I/R injury, and have thus been implicated in the pathogenesis of, for example, myocardial infarction, stroke, and acute tubular necrosis. Additionally, neutrophils have been shown to directly injure vascular endothelial cells and are one of the cell types contributing to the formation of glomerular crescents in ANCA-associated vasculitis.
In any event, despite extensive research into neutrophil activation and priming, and the role of neutrophils in inflammatory processes, current therapies directed toward inhibiting detrimental neutrophil-mediated inflammation are less than sufficient. None of the known therapeutic agents or treatment modalities provide a sufficient therapeutic approach whereby neutrophil exocytosis can be inhibited, and undesirable neutrophil-mediated inflammation and subsequent cellular and tissue damage can be reduced.